Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes in supercritical carbon dioxide

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes was performed in supercritical CO(2) and liquid CO(2) at 308.2 and 333.2 K at pressures of 6.1 and 10.1 MPa. The enzymatic activity of coated alpha-chymotrypsin complexes for dipeptides synthesis at 10.1 MPa in supercritical CO(2) (SC-CO(2)) was higher than that in a liquid CO(2) and ethyl acetate solution at 6.1 MPa. The behavior of alpha-chymotrypsin in SC-CO(2) was similar to that in liquid ethyl acetate. And increasing the pressure and temperature increased the maximum conversion and the enzymatic reaction rate in SC-CO(2). Furthermore, the control of the water content in the reaction media had a dominant effect on the enzymatic activity. The maximum conversion for the dipeptide synthesis by the surfactant-coated alpha-chymotrypsin was obtained at 4% water content. The alpha-chymotrypsin complexes exhibited a higher enzymatic activity than native alpha-chymotrypsin in SC-CO(2). The nonionic surfactants l-glutamic acid dialkyl ester ribitol amide and sorbitan monostearate were more favored than the anionic surfactant sodium bis(2-ethylhexyl)sulfosuccinate.     

The effect of plasma potassium in determining normal rates of excretion of potassium in dogs.

Doses of 5-15 mmol KCl or KHCO3 (less than the daily intake in food) given by stomach tube or intravenous infusion, produced increases in plasma K and in K excretion, the time delay between change in plasma K and rate of excretion being minimal. Without doses of K salts in control experiments, plasma K concentration was about 4 mmol/1 and K excretion about 5 mumol/min. After doses of KCl or KHCO3, plasma K and rate of excretion of K both increased, increase of 0-5 mmol/1 in plasma K being associated with an increase of about 35 mumo1/min in K excretion. Increased excretion of K was accompanied by a small increase in Na excretion. Excretion of both C1 and HCO3 increased, C1 more after HCO3 more after KHCO3. The results indicate that within normal ranges, plasma K is an important factor determining the rate of excretion of K.     

A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.     

Salt hydrates buffer water activity during chymotrypsin-catalysed peptide synthesis.

Chymotrypsin (EC 3.4.21.1) powder suspended in hexane in the presence of Na2CO3.10H2O is a good catalyst for peptide synthesis. The salt hydrate releases water to fix the thermodynamic water activity of the system in accord with its dissociation pressure. Salt hydrates can be useful to buffer water activity in mainly organic enzyme reaction mixtures at a value permitting activity of the catalyst while minimising hydrolytic side reactions.     

Evidence for the Calvin cycle and hexose monophosphate pathway in Thiobacillus ferrooxidans

The enzymes of the Calvin reductive pentose phosphate cycle and the hexose monophosphate pathway have been demonstrated in cell-free extracts of Thiobacillus ferrooxidans. This, together with analyses of the products of CO(2) fixation in cell-free systems, suggests that these pathways are operative in whole cells of this microorganism. Nevertheless, the amount of CO(2) fixed in these cell-free systems was limited by the type and amount of compound added as substrate. The inability of cell extracts to regenerate pentose phosphates and to perpetuate the cyclic fixation of CO(2) is partially attributable to low activity of triose phosphate dehydrogenase under the experimental conditions found to be optimal for the enzymes involved in the utilization of ribose-5-phosphate or ribulose-1,5-diphosphate as substrate for CO(2) incorporation. With the exception of ribulose-1,5-diphosphate, all substrates required the addition of adenosine triphosphate (ATP) or adenosine diphosphate (ADP) for CO(2) fixation. Under optimal conditions, with ribose-5-phosphate serving as substrate, each micromole of ATP added resulted in the fixation of 1.5 mumoles of CO(2), whereas each micromole of ADP resulted in 0.5 mumole of CO(2) fixed. These values reflect the activity of adenylate kinase in the extract preparations. The K(m) for ATP in the phosphoribulokinase reaction was 0.91 x 10(-3)m. Kinetic studies conducted with carboxydismutase showed K(m) values of 1.15 x 10(-4)m and 5 x 10(-2)m for ribulose-1,5-diphosphate and bicarbonate, respectively.     

Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).     

Effect of dimethylsulfoxide on ATP synthesis by mitochondrial soluble F1-ATPase

F1-ATPase isolated from bovine heart mitochondria catalyzes the synthesis of enzyme-bound ATP from externally added ADP and Pi in the presence of dimethylsulfoxide (DMSO) (Sakamoto, J. & Tonomura, Y. (1983) J. Biochem. 93, 1601-1614). When the concentration of DMSO in the reaction medium was decreased from 40% to 10% (w/v), the maximal amount of ATP formed decreased from 0.50 to 0.14 mol/mol F1 and the Pi concentration required for the half-maximal amount of ATP formed increased from 0.7 to 11 mM. On the other hand, the ADP concentration required for the half-maximal value and the rate of ATP formation were unaffected by the decrease in the DMSO concentration. These results suggest that DMSO increases the affinity of F1 and Pi and shifts the equilibrium from the enzyme-ADP-Pi complex to the enzyme-ATP complex during the ATP synthesis.     

Revisiting the effect of water content on enzymatic peptide synthesis in non-aqueous medium

Enzymatic acyl-transfer reaction in organic medium competes with the hydrolytic side reaction depending on the water content. The effect of water content on aminolysis activity of -chymotrypsin for the synthesis of Bz-Tyr-Val-NH₂ in acetonitrile was examined under the conditions which were devoid of the hydrolytic deacylation. Excess H-Val-NH₂ (880 mM) was employed to keep the hydrolysis negligible. The aminolysis rate increased abruptly between 4 and 5% (v/v) water but a further increase in the water content did not affect the reaction rate. This suggests that water added more than 5% (v/v) does not enhance intrinsic enzyme activity but acts only as a nucleophile for the hydrolytic deacylation.     

Whole body and hepatic cholesterol synthesis rates in the guinea-pig: effect of dietary fat quality

The effects of polyunsaturated, monounsaturated and saturated dietary fat on total and hepatic cholesterol synthesis were studied in the guinea-pig. Male Hartley guinea-pigs were fed semi-synthetic diets containing 7.5% (w/w) of either corn oil (CO), olive oil (OL) or lard for a period of 5 weeks and rates of endogenous cholesterol synthesis were determined from the incorporation of [3H]water into digitonin-precipitable sterols (DPS) and by measurement of sterol balance. In addition, total and expressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were determined in hepatic microsomes. Rates of whole body cholesterol synthesis determined by incorporation of [3H]water into DPS were significantly lower for guinea-pigs on the CO diet with values of 18.7 +/- 1.8 mumol/h (n = 4) vs. 26.7 +/- 4.8 and 24.6 +/- 1.8 mumol/h for animals on the OL (n = 4) and lard (n = 3) diets (P less than 0.001), respectively. Hepatic cholesterol synthesis rates were significantly decreased in animals on the OL diet, whether determined from incorporation of [3H]water into DPS or by analysis of HMG-CoA reductase activity. Hepatic total and free cholesterol levels were not different for animals on the three dietary fats; however, cholesteryl ester levels were 35% lower in guinea-pigs fed the lard diet (P less than 0.02). Sterol balance measurements indicated that whole body cholesterol synthesis rates were not affected by dietary fat quality (51.9 +/- 12.2, 42.8 +/- 7.6 and 51.2 +/- 20.2 mg/kg per day for animals on the CO, OL and lard diets, respectively). This is in striking contrast to the observed reduction in cholesterol synthesis rates for animals on the polyunsaturated CO diet as determined by incorporation of [3H]water into DPS. One possible explanation for the discrepancy between the sterol balance and [3H]water incorporation data is a polyunsaturated fat-mediated effect on energy utilization, which affects the equilibration of NADPH with the body water pool such that the [3H]NADPH has a lower specific activity than body [3H]water.     

Mechanism of mitochondrial carbamoyl-phosphate synthetase: synthesis and properties of active CO2, precursor of carbamoyl phosphate.

This paper demonstrates the formation of "active CO2" (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase. Absence of ammonia is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. Adenosine triphosphate (ATP) and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 = 0.75 s at 24 degrees C at neutral pH). In the absence of ammonia, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The "apparent KM'S" are approximately equal to those found for the adenosine triphosphate (ATPase) activity of the enzyme. The maximum level of CO2-P is limited by the amount of enzyme, and approximates 4 mol of intermediate/mol of enzyme. The unprotonated form of ammonia seems to be the species reacting with CO2-P to produce CP. The reaction of CO2-P and NH3 is very fast (rate constant kn = 8 x 10(4) M-1 S-1) and does not consume free ATP. Therefore, the 2 mol of ATP necessary for CP synthesis binds or reacts with the enzyme and/or CO2 prior to reaction with NH3. The reaction of CO2-P with NH3 also takes place in acetone under conditions at which the enzyme is not active, suggesting little or no assistance from enzyme catalysis or that a part of the catalytic site is "frozen" by the solvent in the active conformation. In the light of these and other findings, a new scheme is proposed for the mechanism of frog liver carbamoyl-phosphate synthetase and some considerations are made on the chemical nature of the intermediate and on the possible evolutionary significance of the reaction of CO2-P with NH3 in acetone.     

Thermodynamic parameters monitoring the equilibrium shift of enzyme-catalyzed hydrolysis/synthesis reactions in favor of synthesis in mixtures of water and organic solvent

The main strategy developed to shift the equilibrium state of a hydrolase-catalyzed hydrolysis/synthesis reaction consists in reducing water activity by addition of organic solvents in the reaction medium. We have used several mixtures of water and 1,4-butanediol, ranging from pure water to pure 1,4-butanediol, to study the hydrolysis/synthesis reaction of the N-Cbz-L-tryptophanyl-glycineamide dipeptide, catalyzed by alpha-chymotrypsin. In the presence of 1,4-butanediol, alpha-chymotrypsin also catalyzed the esterification reaction between this diol and N-Cbz-L-tryptophan; this ester hydrolysis/synthesis reaction has thus also been examined. The dipeptide and ester equilibrium concentrations increase when the water content of the reaction medium is decreased. Using our experimental data, we have determined the equilibrium constants of the hydrolysis/synthesis equilibria involving the nonionized forms of the protected amino acids, the estimated values of which are Ksp = 8 10(5) for the dipeptide and Kse = 78 for the ester respectively. They are true thermodynamic equilibrium constants, each related to a single, well-defined reaction equilibrium and with water activity being taken into account. If an organic solvent is added to the reaction medium these equilibria can be shifted towards synthesis by decreasing the water activity but also by modifying the ionization/neutralization equilibrium constant of the ionizable groups. These two effects depend both on the water content and on the nature of the organic solvent used, and, in particular, on its dielectric constant. Because of the importance of this parameter in our study, we discuss using it as an indicator to select an appropriate organic solvent to perform an enzyme-catalyzed synthesis.     

Urease-catalyzed urea synthesis.

The equilibrium of hydrolytic reactions can be shifted toward condensation by carrying out the reaction at low water concentration. The rate and yield of urease-catalyzed urea synthesis from (NH₄)₂CO₃ or NH₄HCO₃ has been examined as a function of water concentration (in mixtures with organic solvents), substrate and H⁺ concentration, and polarity of the nonaqueous component of the solvent. Similar effects of organic solvents are observed on the reaction rate in both directions; the results suggest that at least in some conditions the reaction proceeds through nonenzymically formed carbamate. The equilibrium concentration of urea, in 50% ($\text{v}\text{v}$) water, varies over 10-fold, depending on the nature of the nonaqueous component of the solvent; nonhydroxylic solvents such as acetone given the highest yield. Solubility measurements suggest that the interactions of the solvent mixtures with (NH₄)₂CO₃ (or carbamate), rather than urea, are responsible for the variations in urea yield. Activities of water and the ionic components of the equilibrium are strongly influenced by the nature of the nonaqueous component of the solvent, as well as its concentration.     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.     

Kinetics of enzymatic solid-to-solid peptide synthesis: intersubstrate compound, substrate ratio, and mixing effects

A systematic study of thermolysin-catalyzed solid-to-solid peptide synthesis using Z-Gln and Leu-NH2 as model substrates was carried out. The aim was to extend the kinetic knowledge of this new reaction system involving highly concentrated substrate mixtures with little water (10% to 20% w/w). Preheating of the substrates, and ultrasonic treatment, as described in the literature, had no significant effect on our system. The formation of a third compound, the salt of the two substrates, was discovered during melting point experiments. This was associated with a very strong dependence of kinetics on the exact substrate ratio (e.g., twofold higher initial rate with 60% Leu-NH2 and 40% Z-Gln than with the equimolar substrate ratio). A model was developed to show how the composition and pH of the liquid phase depends on the substrate ratio, and seemed to explain the experimental rates. In addition, the influences of different mixing and water distribution methods are described. Finally, we can now summarize the major effects of the reaction system as a starting point for further research and scale-up studies.     

Protease-catalyzed synthesis of a precursor dipeptide, Z-Asp-Val-NH2 of thymopentin, in organic solvents

The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide, Z-Asp-Val-NH(2) of thymopentin (TP-5), in organic solvents was studied. Z-Asp-OMe and Val-NH(2) were used as the acyl donor and the nucleophile, respectively. An industrial alkaline protease alcalase was used to catalyze the synthesis of the target dipeptide in water-organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-NH(2). The optimum conditions using alcalase as the catalyst are pH 10.0, 35 degrees C, in acetonitrile/Na(2)CO(3)-NaHCO(3) buffer system (9:1, V/V), reaction time 5 h, with a yield of 63%. The dipeptide product was confirmed by LC- MS.     

Improvement of enzymatic synthesis yields of flavour acetates: The example of the isoamyl acetate

Summary Isoamyl acetate synthesis was chosen as a model to improve flavour acetate yields by optimising the enzymatic reaction. Alcohol:acid molar ratio, temperature, water content and amount of enzyme effects were analyzed. The optimum values were respectively 4, 45°C, 0,1% (w/v) and 0,5 g. In these conditions, the synthesis yield reached 80 % after 24 h of reaction and was found 15 times greater than those already reported in the literature.     

Oil formation from glucose with formic acid and cobalt catalyst in hot-compressed water

Liquefaction of glucose into oil was examined in hot-compressed water at 300 degrees C and 30 or 60 min in a tumbling batch reactor. The effects of alkali (KHCO(3)), a hydrogenating agent (HCO(2)H), and a cobalt catalyst (Co(3)O(4)) were studied. Also the combinations of these additives were investigated. HCO(2)H and KHCO(3) showed a positive effect on oil formation. Co(3)O(4) was found to be an advantageous additive as well, increasing the oil formation from glucose, but the stability of this catalyst under reaction conditions was quite low.     

The role of pyruvate ferredoxin oxidoreductase in pyruvate synthesis during autotrophic growth by the Wood-Ljungdahl pathway

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO(2). The catalytic proficiency of this enzyme for the reverse reaction, pyruvate synthase, is poorly understood. Conversion of acetyl-CoA to pyruvate links the Wood-Ljungdahl pathway of autotrophic CO(2) fixation to the reductive tricarboxylic acid cycle, which in these autotrophic anaerobes is the stage for biosynthesis of all cellular macromolecules. The results described here demonstrate that the Clostridium thermoaceticum PFOR is a highly efficient pyruvate synthase. The Michaelis-Menten parameters for pyruvate synthesis by PFOR are: V(max) = 1.6 unit/mg (k(cat) = 3.2 s(-1)), K(m)(Acetyl-CoA) = 9 micrometer, and K(m)(CO(2)) = 2 mm. The intracellular concentrations of acetyl-CoA, CoASH, and pyruvate have been measured. The predicted rate of pyruvate synthesis at physiological concentrations of substrates clearly is sufficient to support the role of PFOR as a pyruvate synthase in vivo. Measurements of its k(cat)/K(m) values demonstrate that ferredoxin is a highly efficient electron carrier in both the oxidative and reductive reactions. On the other hand, rubredoxin is a poor substitute in the oxidative direction and is inept in donating electrons for pyruvate synthesis.     

Prebiotic synthesis in atmospheres containing CH4, CO,and CO2

The prebiotic synthesis of organic compounds using a spark discharge on various simulated primitive earth atmospheres at 25 degrees C has been studied. Methane mixtures contained H2 + CH4 + H2O + N2 + NH3 with H2/CH4 molar ratios from 0 to 4 and pNH3 = 0.1 torr. A similar set of experiments without added NH3 was performed. The yields of amino acids (1.2 to 4.7% based on the carbon) are approximately independent of the H2/CH4 ratio and whether NH3 was present, and a wide variety of amino acids are obtained. Mixtures of H2 + CO + H2O + N2 and H2 + CO2 + H2O + N2, with and without added NH3, all gave about 2% yields of amino acids at H2/CO and H2/CO2 ratios of 2 to 4. For a H2/CO2 ratio of 0, the yield of amino acids is extremely low (10(-3)%). Glycine is almost the only amino acid produced from CO and CO2 model atmospheres. These results show that the maximum yield is about the same for the three carbon sources at high H2/carbon ratios, but that CH4 is superior at low H2/carbon ratios. In addition, CH4 gives a much greater variety of amino acids than either CO or CO2. If it is assumed that an abundance of amino acids more complex than glycine was required for the origin of life, then these results indicate the requirement for CH4 in the primitive atmosphere.     

Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica

The effect of solvents and solvent mixtures on the synthesis of myristic acid esters of different carbohydrates with an immobilized lipase from C. antarctica was investigated. The rate of myristyl glucose synthesized by the enzyme was increased from 3.7 to 20.2 micromol min(-1) g(-1) by changing the solvent from pure tert-butanol to a mixture of tert-butanol:pyridine (55:45 v/v), by increasing the temperature from 45 degrees C to 60 degrees C, and by optimizing the relative amounts of glucose, myristic acid, and the enzyme preparation. Addition of more than 2% DMSO to the tert-butanol:pyridine system resulted in a reduction of enzyme activity. Lowering the water content of the enzyme preparation below 0.85% (w/w) resulted in significant decreases in enzyme activity, while increasing the water content up to 2.17% (w/w) did not significantly affect the enzyme activity. The highest yields of myristyl glucose were obtained when an excess of unsolubilized glucose was present in the reaction system. In this case, all of the initially solubilized and a significant amount of the initially unsolubilized glucose was converted to the ester within 24 h of incubation, resulting in a myristyl glucose concentration of 34 mg/mL(-1). Myristic acid esters of fructose (22.3 micromol min(-1) g(-1)), alpha-D-methyl-glucopyranoside (26.9 micromol min(-1) g(-1)) and maltose (1.9 micromol min(-1) g(-1)) could also be prepared using the tert-butanol:pyridine solvent system. No synthesis activity was observed with maltotriose, cellobiose, sucrose, and lactose as substrate.